This invention relates generally to the field of cell growth and, more particularly, to using polyelectrolyte complex films to coat surfaces to enhance or inhibit cell growth, adhesion, and differentiation.
In order for cells to adhere and grow on a substrate, the interface between the substrate and the cell growth medium must possess an appropriate combination of physical and chemical properties. Control over the surface of substrate provides for control over cell adhesion. Advantageous properties imparted by a surface range from the complete rejection of any cell adhesion or growth, to cell adhesion without growth, to cell adhesion, growth, and differentiation. The property desired depends on the end-use of the substrate. For example, articles implanted in vivo, such as stents, catheters, and artificial organs, preferably do not induce biochemical processes that lead to scarring and/or rejection of said article. These implants may be advantageously coated with thin films that render them biocompatible. Alternatively, some applications, especially those in tissue engineering, require substrates that encourage the growth, differentiation, and proliferation of cells. A strategy for modifying the cell adhesion and cell growth properties of surfaces is needed.
Polyelectrolytes are macromolecules comprising a plurality of charged repeat units. Amorphous complexes may be formed by contacting solutions of polyelectrolytes bearing opposite charges. The driving force for association, or complexation, of polyelectrolytes is multiple ion pairing between oppositely charged repeat units on different molecules.
Recently, thin films of polyelectrolyte complexes have been prepared using polyelectrolytes which are alternately deposited on a substrate or substratum. See Decher and Schlenoff, Eds., Multilayer Thin Films—Sequential Assembly of Nanocomposite Materials, Wiley-VCH, Weinheim (2003); Decher, Science, 277, 1232 (1997). Decher and Hong (U.S. Pat. No. 5,208,111) disclose a method for a buildup of multilayers by alternating dipping, i.e., cycling a substrate between two reservoirs containing aqueous solutions of polyelectrolytes of opposite charge, with an optional rinse step in polymer-free solution following each immersion. Each cycle adds a layer of polymer via ion pairing forces to the oppositely-charged surface and reverses the surface charge thereby priming the film for the addition of the next layer. Films prepared in this manner tend to be uniform, follow the contours and irregularities of the substrate, and are typically between about 10 nm and about 10,000 nm thick. The thickness of a film depends on many factors, including the number of layers deposited, the ionic strength of the solutions, the types of polymers, the deposition time, the solution pH, the temperature, and the solvent used. Although studies have shown that substantial interpenetration of the individual polymer layers results in little composition variation over the thickness of a film, such polymer thin films are, nevertheless, referred to as polyelectrolyte multilayers (PEMUs).
Surface modification using polyelectrolyte multilayers to develop biocompatible materials has been attracting attention lately due to the ease of synthesis and cost-effectiveness of the layer-by-layer technique. See Decher, G., Schlenoff, J. B. Multilayer Thin Films—Sequential Assembly of Nanocomposite Materials; Wiley-VCH: Weinheim, Germany, 2003. Surface properties ranging from hydrophobic to hydrophilic, charged to uncharged, and smooth to rough can be generated using a variety of parameters including the chemical nature of the polyelectrolytes and the pH, ionic strength, and temperature used for multilayer synthesis. Because proteins play an important role in the adhesion, spreading, and growth of cells, considerable effort has been expended in developing polyelectrolyte thin films with properties that make the surface adhesive or resistant to protein adsorption. See Muller, M.; Rieser, T.; Kothe, M.; Kessler, B.; Brissova, M.; Lunkwitz, K. Macromol. Symp. 1999, 145, 149, Muller, M.; Brissova, M.; Rieser, T.; Powers, A. C.; Lunkwitz, K. Mat. Sci. Eng. C-Bio. S. 1999, 8-9, 163, Muller, M.; Rieser, T.; Lunkwitz, K.; Meier-Haack, J. Macromol. Rapid. Comm. 1999, 20, 607, Ladam, G.; Gergely, C.; Senger, B.; Decher, G.; Voegel, J. C.; Schaaf, P.; Cuisinier, F. J. G. Biomacromolecules 2000, 1, 674, Ladam, G.; Schaaf, P.; Cuisinier, F. J. G.; Decher, G.; Voegel, J. C. Langmuir 2001, 17, 878, and Salloum, D. S.; Schlenoff, J. B. Biomacromolecules 2004. Although an understanding of PEMU-protein adsorption is necessary to intelligently engineer cell-biomaterial interaction, it is difficult to predict PEMU-cell biocompatibility from simple measurements of protein adsorption. See Han, D. K.; Ryu, G. H.; Park, K. D.; Jeong, S. Y.; Kim, Y. H.; Min, B. G. Journal of Biomaterials Science-Polymer Edition 1993, 4, 401, Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336, and Mendelsohn, J. D.; Yang, S. Y.; Hiller, J.; Hochbaum, A. I.; Rubner, M. F. Biomacromolecules 2003, 4, 96. Recent investigations using cultured cells revealed PEMU properties important for cell biocompatibility. See Ito, Y.; Chen, G. P.; Imanishi, Y. Bioconjugate Chemistry 1998, 9, 277, Chluba, J.; Voegel, J. C.; Decher, G.; Erbacher, P.; Schaaf, P.; Ogier, J. Biomacromolecules 2001, 2, 800, Tryoen-Toth, P.; Vautier, D.; Haikel, Y.; Voegel, J. C.; Schaaf, P.; Chluba, J.; Ogier, J. Journal of Biomedical Materials Research 2002, 60, 657, Richert, L.; Lavalle, P.; Vautier, D.; Senger, B.; Stoltz, J. F.; Schaaf, P.; Voegel, J. C.; Picart, C. Biomacromolecules 2002, 3, 1170, Boura, C.; Menu, P.; Payan, E.; Picart, C.; Voegel, J. C.; Muller, S.; Stoltz, J. F. Biomaterials 2003, 24, 3521, Elbert, D. L.; Herbert, C. B.; Hubbell, J. A. Langmuir 1999, 15, 5355, and Serizawa, T.; Yamaguchi, M.; Akashi, M. Biomacromolecules 2002, 3, 724. These investigations have demonstrated that surfaces can be rendered cytophilic or cytophobic by embedding or attaching protein or peptides to the multilayer and by tuning the pH used for multilayer buildup. See Jessel, N.; Atalar, F.; Lavalle, P.; Mutterer, J.; Decher, G.; Schaaf, P.; Voegel, J. C.; Ogier, J. Adv. Mater. 2003, 15, 692 and Berg, M. C.; Yang, S. Y.; Hammond, P. T.; Rubner, M. F. Langmuir 2004, 20, 1362. Other modifications such as chemical cross-linking have improved the PEMU stability and cell adhesion. See Richert, L.; Boulmedais, F.; Lavalle, P.; Mutterer, J.; Ferreux, E.; Decher, G.; Schaaf, P.; Voegel, J. C.; Picart, C. Biomacromolecules 2003. Certain surfaces such as polysaccharide films made by layer-by-layer buildup have been investigated for use as antimicrobial coatings and bioactive endovascular stent coatings. See Richert, L.; Lavalle, P.; Payan, E.; Shu, X. Z.; Prestwich, G. D.; Stoltz, J. F.; Schaaf, P.; Voegel, J. C.; Picart, C. Langmuir 2004, 20, 448 and Thierry, B.; Winnik, F. M.; Merhi, Y.; Silver, J.; Tabrizian, M. Biomacromolecules 2003, 4, 1564. In fact, polyelectrolyte complexes have a long history of use in preparing bioinert surfaces. See Tsuchida, E.; Abe, K. Advances in Polymer Science 1982, 45, 1, Philippe, B.; Dautzenberg, H.; Linow, K. J.; Kotz, J.; Dawydoff, W. Progress in Polymer Science 1989, 14, 91, and Petrak, K. Journal of Bioactive and Compatible Polymers 1993, 8, 178.
The surface properties of endovascular stents may play an important role in the process of restenosis. During restenosis, vascular smooth muscle cells migrate to cover implanted stents, often building layers of tissue that cause occlusion of the blood flow. See Indolfi, C.; Mongiardo, A.; Curcio, A.; Torella, D. Trends in Cardiovascular Medicine 2003, 13, 142. In evaluating biocompatibility, therefore, it is important to understand how smooth muscle cells interact with PEMU surfaces. Smooth muscle cells are capable of alternating between a ‘contractile’ phenotype, characterized by a non-motile cell type that possesses both a contractile smooth muscle cytoskeleton and a non muscle cytoskeleton for cell support, and a ‘synthetic’ phenotype that is motile and possesses a non-muscle cytoskeleton used for cell support and cell motility. See Worth, N. F.; Rolfe, B. E.; Song, J.; Campbell, G. R. Cell Motility and the Cytoskeleton 2001, 49, 130 and Halayko, A. J.; Solway, J. Journal of Applied Physiology 2001, 90, 358. The two cell phenotypes can be readily distinguished by the cell shape, stability of adhesion, and organization of the underlying cytoskeleton structures.